High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes

被引:38
作者
Seegers, Nicole [1 ]
van Doornmalen, Antoon M. [1 ]
Uitdehaag, Joost C. M. [1 ]
de Man, Jos [1 ]
Buijsman, Rogier C. [1 ]
Zaman, Guido J. R. [1 ]
机构
[1] Netherlands Translat Res Ctr BV, NL-5342 CC Oss, Netherlands
关键词
indoleamine; 2; 3-dioxygenase; tryptophan; N-formyl kynurenine; high-throughput screening; cancer immunotherapy; neurodegenerative disease; HUMAN INDOLEAMINE 2,3-DIOXYGENASE; TUMORAL IMMUNE RESISTANCE; INHIBITION; CANCER; DEGRADATION; MECHANISM; CELLS; DIOXYGENASE; PREVENTION; KYNURENINE;
D O I
10.1177/1087057114536616
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.
引用
收藏
页码:1266 / 1274
页数:9
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